Method for detecting cytosine methylation in DNA samples

ABSTRACT

Described is a method for detecting 5-methylcytosine in genomic DNA samples. First, a genomic DNA from a DNA sample is chemically converted with a reagent, 5-methylcytosine and cytosine reacting differently, and the pretreated DNA is subsequently amplified using a polymerase and at least one primer. In the next step, the amplified genomic DNA is hybridized to at least one oligonucleotide, forming a duplex, and said oligonucleotide is elongated by at least one nucleotide, the nucleotide carrying a detectable label, and the elongation depending on the methylation status of the specific cytosine in the genomic DNA sample. In the next step, the elongated oligonucleotides are analyzed for the presence of the label.

The present invention relates to a method for detecting 5-methylcytosinein genomic DNA samples.

The levels of observation that have been well studied by themethodological developments of recent years in molecular biology are thegenes themselves, the translation of genes into RNA, and the resultingproteins. The question of which gene is switched on at which point inthe course of the development of an individual, and how the activationand inhibition of specific genes in specific cells and tissues arecontrolled is correlatable to the degree and character of themethylation of the genes or of the genome.

The present invention describes a method for detecting the methylationstate of genomic DNA samples. The method can, at the same time, also beused for detecting point mutations and single nucleotide polymorphisms(SNPs).

5-methylcytosine is the most frequent covalently modifiable base in theDNA of eukaryotic cells. It plays a role, for example, in the regulationof the transcription, in genetic imprinting, and in tumorigenesis.Therefore, the identification of 5-methylcytosine as a part of geneticinformation is of considerable interest. However, 5-methylcytosinepositions cannot be identified by sequencing since 5-methylcytosine hasthe same base pairing behavior as cytosine. Moreover, the epigeneticinformation carried by the 5-methylcytosines is completely lost during aPCR amplification.

A relatively new and now the most frequently used method for analyzingDNA for 5-methylcytosine is based on the specific reaction of bisulfitewith cytosine which, upon subsequent alkaline hydrolysis, is convertedto uracil which corresponds to thymidine in its base pairing behavior.However, 5-methylcytosine remains unmodified under these conditions.Consequently, the original DNA is converted in such a manner thatmethylcytosine, which originally cannot be distinguished from cytosinein its hybridization behavior, can now be detected as the only remainingcytosine using “normal” molecular biological techniques, for example, byamplification and hybridization or sequencing. All these techniques arebased on base pairing which is now taken full advantage of. The PriorArt is defined in terms of sensitivity by a method which encloses theDNA to be analyzed in an agarose matrix, thus preventing the diffusionand renaturation of the DNA (bisulfite reacts only on single-strandedDNA), and which replaces all precipitation and purification steps withfast dialysis (Olek, A. et al, Nucl. Acids. Res. 1996, 24, 5064-5066).Using this method, it is possible to analyze individual cells, whichillustrates the potential of the method. Up to now, however, onlyindividual regions of a length of up to approximately 3000 base pairsare analyzed; a global analysis of cells for thousands of possiblemethylation analyses is not possible. However, this method cannotreliably analyze very small fragments from small sample quantitieseither. These are lost in spite of the diffusion protection by thematrix.

An overview of the further known possibilities of detecting5-methylcytosines can be gathered from the following survey article:Rein, T., DePamphilis, M. L., Zorbas, H., Nucleic Acids Res. 1998, 26,2255.

Up to now, the bisulfite technology is only used in research with fewexceptions (e.g., Zeschnigk M. et al, Eur J Hum Genet. 1997, 5, 94-98).Always, however, short specific fragments of a known gene are amplifiedsubsequent to a bisulfite treatment and either completely sequenced(Olek, A. and Walter, J., Nat. Genet. 1997, 17, 275-276) or individualcytosine positions are detected by a primer extension reaction(Gonzalgo, M. L., and Jones, P. A., Nucl. Acids Res. 1997, 25,2529-2531, WO 9500669) or by an enzymatic digestion (Xiong, Z. andLaird, P. W., Nucl. Acids. Res. 1997, 25, 2532-2534). In addition, thedetection by hybridization has also been described (Olek et al., WO 9928498).

Further publications dealing with the use of the bisulfite technique formethylation detection in individual genes are: Xiong, Z. and Laird, P.W. (1997), Nucl. Acids Res. 25, 2532; Gonzalgo, M. L. and Jones, P. A.(1997), Nucl. Acids Res. 25, 2529; Grigg, S, and Clark, S. (1994),Bioassays 16, 431; Zeschnik, M. et al. (1997), Human Molecular Genetics6, 387; Teil, R. et al. (1994), Nucl. Acids Res. 22, 695; Martin, V. etal. (1995), Gene 157, 261; WO 97 46705 and WO 95 15373.

An overview of the Prior Art in oligomer array manufacturing can begathered from a special edition of Nature Genetics (Nature GeneticsSupplement, Volume 21, January 1999), published in January 1999, andfrom the literature cited there.

There are different methods for immobilizing DNA. The best-known methodis the fixed binding of a DNA which is functionalized with biotin to astreptavidin-coated surface (Uhlen, M. et al. 1988, Nucleic Acids Res.16, 3025-3038). The binding strength of this system corresponds to thatof a covalent chemical bond without being one. To be able to covalentlybind a target DNA to a chemically prepared surface, a correspondingfunctionality of the target DNA is required. DNA itself does not possessany functionalization which is suitable. There are different variants ofintroducing a suitable functionalization into a target DNA: twofunctionalizations which are easy to handle are primary aliphatic aminesand thiols. Such amines are quantitatively converted withN-hydroxysuccinimide esters, and thiols react quantitatively with alkyliodides under suitable conditions. A difficulty consists in introducingsuch a functionalization into a DNA. The simplest variant is theintroduction via a primer of a PCR. Disclosed variants use 5′-modifiedprimers (NH₂ and SH) and a bifunctional linker.

An essential component of the immobilization on a surface is itsconstitution. Systems described up to now are mainly composed of siliconor metal. A further method of binding a target DNA is based on the useof a short recognition sequence (e.g., 20 bases) in the target DNA forhybridization to a surface-immobilized oligonucleotide. Enzymaticvariants for introducing chemically activated positions in a target DNAhave been described as well. In this case, a 5′-NH₂-functionalization iscarried out enzymatically on a target DNA.

For scanning an immobilized DNA array, fluorescently labeled probes haveoften been used. Particularly suitable for fluorescence labeling is thesimple attachment of Cy3 and Cy5 dyes to the 5′-OH of the specificprobe. The detection of the fluorescence of the hybridized probes iscarried out, for example via a confocal microscope. Cy3 and Cy5 dyes,besides many others, are commercially available.

More recent methods for detecting mutations are specified in thefollowing:

Worth mentioning as a special case of sequencing is the single-baseprimer extension (Genetic Bit Analysis) (Head, S R., Rogers, Y H.,Parikh K., Lan, G., Anderson, S., Goelet, P., Boycejacino M T., NucleicAcids Research. 25(24): 5065-5071, 1997; Picoult-Newberg, L., GenomeRes. 9(2): 167-174, 1999). A combined amplification and sequencing isdescribed in US-A15928906 where a base-specific termination on matrixmolecules is used. A further method uses a ligase/polymerase reactionfor identifying nucleotides (U.S. Pat. No. 5,952,174).

Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI) isa very efficient development for the analysis of biomolecules (Karas, M.and Hillenkamp, F. (1988), Laser desorption ionization of proteins withmolecular masses exceeding 10000 daltons. Anal. Chem. 60: 2299-2301). Ananalyte is embedded in a light-absorbing matrix. By a short laser pulse,the matrix is evaporated, thus transporting the analyte molecule intothe vapor phase in an unfragmented manner. The analyte is ionized bycollisions with matrix molecules. An applied voltage accelerates theions into a field-free flight tube. Due to their different masses, theions are accelerated at different rates. Smaller ions reach the detectorsooner than bigger ones.

MALDI is ideally suited to the analysis of peptides and proteins. Theanalysis of nucleic acids is somewhat more difficult (Gut, I. G. andBeck, S. (1995), DNA and Matrix Assisted Laser Desorption IonizationMass Spectrometry. Molecular Biology Current Innovations and FutureTrends 1: 147-157.). The sensitivity for nucleic acids is approximately100 times worse than for peptides and decreases disproportionally withincreasing fragment size. For nucleic acids having a multiply negativelycharged backbone, the ionization process via the matrix is considerablyless efficient. For MALDI, the selection of the matrix plays aneminently important role. For the desorption of peptides, several veryefficient matrixes have been found which produce a very finecrystallization. For DNA, there are currently several matrixes in use,however, this has not reduced the difference in sensitivity. Thedifference in sensitivity can be reduced by chemically modifying the DNAin such a manner that it becomes more similar to a peptide.Phosphorothioate nucleic acids in which the usual phosphates of thebackbone are substituted by thiophosphates can be converted into acharge-neutral DNA using simple alkylation chemistry (Gut, I. G. andBeck, S. (1995), A procedure for selective DNA alkylation and detectionby mass spectrometry. Nucleic Acids Res. 23: 1367-1373). The coupling ofa charge tag to this modified DNA results in an increase in sensitivityby the same amount as that found for peptides. A further advantage ofcharge tagging is the increased stability of the analysis againstimpurities which makes the detection of unmodified substratesconsiderably more difficult.

Genomic DNA is obtained from DNA of cell, tissue or other test samplesusing standard methods. This standard methodology is found in referencessuch as Fritsch and Maniatis eds., Molecular Cloning: A LaboratoryManual, 1989.

Mutualities between promoters consist not only in the occurrence ofTATA- or GC-boxes but also for which transcription factors they possessbinding sites and at what distance these are located from each other.The existing binding sites for a specific protein do not matchcompletely in their sequence but conserved sequences of at least 4 basesare found which can still be elongated by inserting wobbles, i.e.,positions at which in each case different bases are located. Moreover,these binding sites are present at specific distances from each other.

However, the distribution of the DNA in the interphase chromatin whichoccupies the largest portion of the nuclear volume is subject to a veryspecial arrangement. Thus, the DNA is attached to the nuclear matrix, afilamentous pattern at the inner side of the nuclear membrane, atseveral locations. These regions are designated as matrix attachmentregions (MAR) or scaffold attachment regions (SAR). The attachment hasan essential influence on the transcription or the replication. TheseMAR fragments have no conserved sequences but to 70% they consist of Aor T, and are located in the vicinity of cis-acting regions whichregulate the transcription in a general manner, and in the vicinity oftopoisomerase II recognition sites.

In addition to promoters and enhancers, further regulatory elements,so-called “insulators”, exist for different genes. These insulators can,for example, inhibit the action of the enhancer on the promotor if theyare located between enhancer and promotor, or else, if located betweenheterochromatin and a gene, can protect the active gene from theinfluence of the heterochromatin. Examples of such insulators include:firstly, so-called “LCR” (locus control regions) consisting of severalsites which are hypersensitive to DNAase I; secondly, certain sequencessuch as SCS (specialized chromatin structures) or SCS′, 350 or 200 bplong, respectively, and highly resistant to degradation by DNAase I, andflanked on both sides with hypersensitive sites (distance in each case100 bp). The protein BEAF-32 binds to scs′. These insulators can belocated on both sides of the gene.

It is the aim of the present invention to provide a method particularlysuitable for concurrently detecting cytosine methylations and SNPs ingenomic DNA samples. In the process, it should preferably be possiblefor a plurality of fragments to be analyzed concurrently.

The aim of the invention is reached by a method for detecting5-methylcytosine in genomic DNA samples, wherein the following steps arecarried out:

(a) a genomic DNA from a DNA sample is chemically converted with areagent, 5-methylcytosine and cytosine reacting differently, thusexhibiting different base pairing behaviors in the DNA duplex subsequentto the reaction;

(b) pretreated DNA is amplified using a polymerase and at least oneoligonucleotide (type A) as a primer;

(c) the amplified genomic DNA is hybridized to at least oneoligonucleotide (type B) having a known sequence of n nucleotides,forming a duplex, said hybridized oligonucleotides of type B, with their3′-ends, partially or completely hybridizing to the positions to beanalyzed with regard to their methylation in the genomic DNA sample;

(d) the oligonucleotide (type B), provided that it has previouslyhybridized with its 3′-terminus to the position to be analyzed withoutmispairings, is elongated by at least one nucleotide by means of apolymerase, at least one nucleotide carrying a detectable label, and theelongation depending on the methylation status of the specific cytosinein the genomic DNA sample;

(e) the elongated oligonucleotides are analyzed for the presence of thelabel.

According to the invention it is preferred that the oligonucleotides(type B) are bonded to a solid phase at defined locations or that theamplificates are bonded to a solid phase at defined locations.

It is further preferred that different oligonucleotide sequences arearranged on a plane solid phase in the form of a rectangular orhexagonal lattice.

According to the invention it is further preferred that the solid phasesurface is composed of silicon, glass, polystyrene, aluminum, steel,iron, copper, nickel, silver, or gold.

It is also preferred that the labels attached to the elongatedoligonucleotides are identifiable at each position of the solid phase atwhich an oligonucleotide sequence is located.

According to the invention it is preferred that at least one primer(type A) is bonded to a solid phase during amplification.

Moreover it can be preferred according to the invention that differentamplificates are arranged on the solid phase in the form of arectangular or hexagonal lattice.

It is particularly preferred that, prior to the amplification, the DNAis treated with a bisulfite solution (=disulfite, hydrogen sulfite).

According to the invention it is particularly preferred that theamplification is carried out by means of the polymerase chain reaction(PCR).

According to the invention it is furthermore preferred that theoligonucleotides of type A either contain only the bases T, A and C orelse the bases T, A and G.

It is further preferred that the labels of the nucleotides arefluorescence labels.

According to the invention it is further preferred that the labels ofthe nucleotides are radionuclides.

It is especially preferred that the labels of the nucleotides aredetachable mass labels which are detected in a mass spectrometer.

According to the invention it is furthermore preferred that theelongated oligonucleotides altogether are detected in the massspectrometer, thus being uniquely labeled by their masses. It is alsoparticularly preferred that in each case one fragment of the elongatedoligonucleotides is detected in the mass spectrometer.

According to the invention it is further preferred that the fragment ofthe elongated oligonucleotide is produced by digestion with one orseveral exo- or endonucleases.

According to the invention it is furthermore preferred that the producedfragments have a single positive or negative net charge for betterdetectability in the mass spectrometer.

According to the invention it is especially preferred that the detectionof the elongated oligonucleotides is carried out and visualized by meansof matrix assisted laser desorption/ionization mass spectrometry (MALDI)or using electron spray mass spectrometry (ESI).

According to the invention a method is preferred wherein the polymerasesare heat-resistant DNA-polymerases.

According to the invention a method is also preferred wherein SNPs arealso detected and visualized in addition to the DNA methylation.

Furthermore a method is preferred wherein the used nucleotides areterminating (type C 2) and/or chain-elongating nucleotides (type C 1).

According to the invention a method is further preferred wherein thenucleotides (type C 1 and C 2) are selected from a group comprisingeither the nucleobases A, T and C or else the bases G and A and T.

According to the invention it is further preferred that theamplification of several DNA segments is carried out in one reactionvessel.

Particularly preferred according to the invention is a method whereinthe genomic DNA is obtained from a DNA sample, sources of DNAcomprising, e.g., cell lines, blood, sputum, stool, urine,cerebral-spinal fluid, tissue embedded in paraffin, histologic objectslides, and all possible combinations thereof.

Finally it is preferred according to the invention that the methylationanalyses of the upper and lower DNA strand is carried out in oneexperiment.

Described is a method for detecting methylcytosine in genomic DNAsamples:

The method includes the amplification, hybridization and elongationreaction of an entire DNA or of a fragment thereof. The method can beused for detecting methylcytosine and, at the same time, also of singlenucleotide polymorphisms (SNPs) and mutations.

The genomic DNA to be analyzed is preferably obtained from usual sourcesof DNA such as cell lines, blood, sputum, stool, urine, cerebral-spinalfluid, tissue embedded in paraffin, histologic object slides, and allpossible combinations thereof.

In the first step of the method, the used DNA is preferably treated withbisulfite (=disulfite, hydrogen sulfite) or else with another chemicalin such a manner that all cytosine bases which are not methylated at the5-position of the base are changed in such a manner that a differentbase results with regard to the base pairing behavior while thecytosines methylated at the 5-position remain unchanged. If bisulfite isused, then an addition takes place at the non-methylated cytosine bases.The subsequent alkaline hydrolysis then gives rise to the conversion ofnon-methylated cytosine nucleobases to uracil.

In the second step of the method, the pretreated DNA is preferablyamplified using a heat-resistant polymerase and at least one primer(type A).

In a particularly preferred variant of the method, the amplification iscarried out with primers of type A by means of the polymerase chainreaction (PCR).

In a preferred variant of the method, the amplification of several DNAfragments is carried out in one reaction vessel. This can either be aso-called “multiplex PCR” in which different primers each producedefined fragments. Different, defined amplifications are carried out inone reaction vessel. In a further, particularly preferred variant of themethod, primers in each case selectively and reproducibly amplifyseveral fragments. This is achieved, for example, in that they bind, forexample, to repetitive elements in the genome. In a particularlypreferred variant of the method, the primers bind to transcriptionfactor binding sites, to promoters or other regulatory elements ingenes. In a particularly preferred variant of the method, theamplification is carried out by elongating primers which are bonded to asolid phase. A multiplex PCR in the broader sense can be carried out inthat different primers are bonded at different, defined locations of asolid phase.

In an, again, preferred variant of the second method step, the solidphase is plane, the different oligonucleotide sequences being arrangedin the form of a rectangular or hexagonal lattice. The result of this isthat the different amplificates are arranged on the solid phase in theform of a rectangular or hexagonal lattice, as well. In this case, asalready described above, several amplificates are directly produced onthe solid phase.

The solid phase surface is preferably composed of silicon, glass,polystyrene, aluminum, steel, iron, copper, nickel, silver, or gold.

In a particularly preferred variant of the method, the oligonucleotidesof type A either contain only bases T, A and C or only bases T, A and G.

In the third method step, the amplified genomic DNA is hybridized to atleast one primer (type B), forming a duplex. The hybridizedoligonucleotides of type B each bind at their 3′-end to the positions tobe analyzed with regard to their methylation in the genomic DNA sample.In this context, two cases can be distinguished: the sequence to beanalyzed is either completely complementary to the primer even at its3′-end; in this case, it is possible to elongate the primer in apolymerase reaction in the next step, or else, the sequence is notcompletely complementary to that of the primer at the 3′-end; in thiscase, the primer cannot be elongated. If a specific CpG-position is tobe analyzed for methylation, then there are two possible conditions.Subsequent to the chemical pretreatment, preferably with bisulfite, a CGoccurs in the case of methylation; if an unmethylated cytosine ispresent, a UG or, subsequent to amplification, a TG ensue. In this case,the experiment is preferably carried out with two different primerswhich give rise to complete complementarity for one of the conditions,respectively, and, consequently, to the possibility of a chainelongation in one of the two possible cases, respectively.

Unless the amplificates are already bonded to the solid phase, then theoligonucleotides which are hybridized to the amplificates can be bondedto a solid phase with their 5′-end, or with another base, or via theirbackbone but not via their 3′-end. Preferably, the binding occurs viathe 5′-end. In a preferred variant, the solid phase is plane, thedifferent oligonucleotide sequences (type B) being arranged in the formof a rectangular or hexagonal lattice.

The solid phase surface is preferably composed of silicon, glass,polystyrene, aluminum, steel, iron, copper, nickel, silver, or gold.

In the fourth method step, the resulting oligonucleotide is elongatedwith a heat-resistant polymerase by at least one up to a maximum of tennucleotides, at least one nucleotide carrying a detectable label. Inthis context, the type of elongation depends on the methylation statusof the specific cytosine in the genomic DNA sample or else also onpossibly existing SNPs, point mutations or deletions, insertions andinversions.

In principle, only terminating oligonucleotides (type C 2) are required.Depending on the sequence, however, chain-elongating oligonucleotidescan be used as well provided that it is possible in the specificsequence context.

In a preferred variant of the method, the used nucleotides areterminating (type C 2) and/or chain-elongating nucleotides (type C 1).In a particularly preferred variant of the method, the nucleobases oftype C1 and/or of type C 2 are selected from a group including bases T,A and C or else bases T, A and G.

The labeling of the elongated oligonucleotides of type B is preferablycarried out via absorbing dyes and/or via chemiluminescence and/or viaradioactive isotopes and/or via fluorescence labels which are introducedvia the nucleotides added in the fourth method step. Also preferred isthe labeling via the molecular mass of the elongated oligonucleotide.The fluorescence label is preferably inserted by a fluorescently labelednucleotide such as Cy5-dCTP.

In the fifth method step, the elongated oligonucleotides are analyzedfor the presence of a label. If a plane solid phase is used, then ananalysis takes place at each location on the solid phase at which,originally, an oligonucleotide was immobilized.

In a particularly preferred variant of the method, the detection of theelongated oligonucleotides is carried out via their fluorescence.

In a preferred variant of the method, fragments of the elongatedoligonucleotide are produced by digestion with one or several exo- orendonucleases.

In a particularly preferred variant of the method, the labels of thenucleotides are detachable mass labels which are detectable in a massspectrometer.

In a particularly preferred variant of the method, detachable masslabels, the elongated oligonucleotides altogether or fragments thereofare detected and visualized on the basis of their unique mass by meansof matrix assisted laser desorption/ionization mass spectrometry(MALDI-MS) or using electron spray mass spectrometry (ESI).

The fragments detected in the mass spectrometer preferably have a singlepositive or negative net charge.

In a particularly preferred variant of the method, SNPs (singlenucleotide polymorphisms) and cytosine methylations are analyzed in oneexperiment.

In a particularly preferred variant of the method, the lower and theupper strand of the DNA sample is analyzed in one experiment subsequentto the chemical pretreatment to ensure an internal experimental control.

A further subject matter of the present invention is a kit containingchemicals and aids for carrying out the bisulfite reaction and/or theamplification, the hybridization, the elongation reaction and/orpolymerases and/or the documentation for carrying out the method.

The following examples illustrate the invention.

EXAMPLE 1

The following example relates to a fragment of exon 23 of the factorVIII gene in which a specific CG-position is to be analyzed formethylation.

In the first step, the fragment is amplified by primers of type A,namely by ATTATGTTGGAGTAGTAGAGTTTAAATGGTT (SEQ.-ID No.: 1) andACTTAACACTTACTATTTAAATCACAACCCAT (SEQ.-ID No.: 2). The amplified DNA ishybridized to an oligonucleotide of type B (for example,GTTGGATGTTGTTGAGAAACG (SEQ.-ID No.: 3)) and elongated in a polymerasereaction with a labeled 2′,3′-didesoxythymidine triphosphat (type C 2).This elongation can only take place if a CG, that is, in the originalgenomic DNA sample, a methylated cytosine was present since otherwise, amispairing at the 3′-end of the primer prevents the polymerase reaction.Thus, the methylation status of the specific cytosine to be analyzeddecides on the elongation of the primer.

For control purposes, the reaction can be carried out with the primerGTTGGATGTTGTTGAGAAATG (SEQ.-ID No.: 4). In this case, the elongationtakes place only if an non-methylated cytosine was present at saidposition in the DNA sample to be analyzed. The labels can, for example,be absorbing dyes such as Megaprime™ for ddTTP or Rediprime II™.

EXAMPLE 2

The following example relates to a fragment of exon 23 of the factorVIII gene in which a specific CG-position is to be analyzed formethylation.

In the first step, the fragment is amplified by primers of type A,namely by ATTATGTTGGAGTAGTAGAGTTTAAATGGTT (SEQ.-ID No.: 1) andACTTAACACTTACTATTTAAATCACAACCCAT (SEQ.-ID No.: 2). The amplified DNA ishybridized to an oligonucleotide of type B (for example,GTTGGATGTTGTTGAGAAACG (SEQ.-ID No.: 3)), which is immobilized to a solidphase surface with its 5′-end, and elongated in a polymerase reactionwith a labeled 2′,3′-didesoxythymidine triphosphat (type C2). Thiselongation can only take place if a CG, that is, in the original genomicDNA sample, a methylated cytosine was present since otherwise, amispairing at the 3′-end of the primer prevents the polymerase reaction.Thus, the methylation status of the specific cytosine to be analyzeddecides on the elongation of the primer.

For control purposes, the reaction can be carried out with the primerGTTGGATGTTGTTGAGAAATG (SEQ.-ID No.: 4). In this case, the elongationtakes place only if an non-methylated cytosine was present at saidposition in the DNA sample to be analyzed. The labels can, for example,be absorbing dyes such as Megaprime™ for ddTTP or Rediprime II™.

EXAMPLE 3

The following example relates to a fragment of exon 23 of the factorVIII gene in which a specific CG-position is to be analyzed formethylation.

In the first step, the fragment is amplified by primers of type A,namely by ATTATGTTGGAGTAGTAGAGTTTAAATGGTT (SEQ.-ID No.: 1) andACTTAACACTTACTATTTAAATCACAACCCAT (SEQ.-ID No.: 2). For thisamplification, DNA treated with bisulphite was incubated for 5 min at96° C., then denaturated by 40 cycles carried out for 55 sec at 96° C.each, incubated for 75 sec at 61.7° C. (annealing) and incubated for 100sec at 72° C. (elongation). In a subsequent reaction (final extension)the reaction mixture is incubated for 15 min at 72° C. The amplified DNAis hybridized to the solid phase immobilized oligonucleotidesAAAAACTACAAAAACTCT (SEQ-ID No.: 5) (spot 1 in FIG. 1) and toAAAACTACGAAAACTCT (SEQ-ID No.: 6 (spot 2 in FIG. 1). The elongationreaction affords 2′-desoxythymidine triphosphate (dTTP, as type C 1),2′-desoxyguanosine triphosphate (dGTP, as type C 1), 2′-desoxyadenosinetriphosphat (dATP, as type C 1), and 2′-desoxycytidine triphosphate(dCTP, as type C 1), fluorescently labeled 2′-desoxycytidinetriphosphate beeing added additionally in a 3:1 ratio (related tonot-labeled 2′-desoxycytidine triphosphate). The reaction mixture,consisting of the amplificate, the desoxydinucleotide-mixture and 10×buffer is incubated for 15 min at 96° C. The Klenow fragment, used as aDNA polymerase herein, is added for the subsequent extension reactionand the reaction mixture is incubated at 37° C. on the solid phaseovernight. As can be gathered from the following figure, the primerextension carried out suggest on the methylation status of the DNA. Acomparison between spot 1 and spot 2, as illustrated in FIG. 1, allows astatement about the methylation status: in this case here the strengthof the signal of spot 1 gives evidence to a non-methylated CpG.

1. A method for detecting 5-methylcytosine in genomic DNA samples,characterized in that the following steps are carried out: (a) a genomicDNA from a DNA sample is chemically converted with a reagent,5-methylcytosine and cytosine reacting differently, thus exhibitingdifferent base pairing behaviors in the DNA duplex subsequent to thereaction; (b) pretreated DNA is amplified using a polymerase and atleast one oligonucleotide (type A) as a primer; (c) the amplifiedgenomic DNA is hybridized to at least one oligonucleotide (type B)having a known sequence of n nucleotides, forming a duplex, saidhybridized oligonucleotides of type B, with their 3′-ends, partially orcompletely hybridizing to the positions to be analyzed with regard totheir methylation in the genomic DNA sample; (d) the oligonucleotide(type B), provided that it has previously hybridized with its3′-terminus to the position to be analyzed without mispairings, iselongated by at least one nucleotide by means of a polymerase, at leastone nucleotide carrying a detectable label, and the elongation dependingon the methylation status of the specific cytosine in the genomic DNAsample; (e) the elongated oligonucleotides are analyzed for the presenceof the label.
 2. The method as recited in claim 1, characterized in thatthe oligonucleotides (type B) are bonded to a solid phase at definedlocations.
 3. The method as recited in claim 1, characterized in thatthe amplificates are bonded to a solid phase at defined locations. 4.The method as recited in claim 2, characterized in that differentoligonucleotide sequences are arranged on a plane solid phase in theform of a rectangular or hexagonal lattice.
 5. The method as recited inclaim 2, characterized in that solid phase surface is composed ofsilicon, glass, polystyrene, aluminum, steel, iron, copper, nickel,silver, or gold.
 6. The method as recited in claim 2, characterized inthat the labels attached to the elongated oligonucleotides areidentifiable at each position of the solid phase at which anoligonucleotide sequence is located.
 7. The method as recited in claim1, characterized in that at least one primer (type A) is bonded to asolid phase during amplification.
 8. The method as recited in claim 1,characterized in that different amplificates are arranged on the solidphase in the form of a rectangular or hexagonal lattice.
 9. The methodas recited in claim 1, characterized in that, prior to theamplification, the DNA is treated with a bisulfite solution (=disulfite,hydrogen sulfite).
 10. The method as recited in claim 1, characterizedin that the amplification is carried out by means of the polymerasechain reaction (PCR).
 11. The method as recited in claim 1,characterized in that the oligonucleotides of type A used in claim 1either contain only the bases T, A and C or else the bases T, A and G.12. The method as recited in claim 1, characterized in that the labelsof the nucleotides are fluorescence labels.
 13. The method as recited inclaim 1, characterized in that the labels of the nucleotides areradionuclides.
 14. The method as recited in claim 1, characterized inthat the labels of the nucleotides are detachable mass labels which aredetected in a mass spectrometer.
 15. The method as recited in claim 1,characterized in that the elongated oligonucleotides altogether aredetected in the mass spectrometer, thus being uniquely labeled by theirmasses.
 16. The method as recited in claim 1, characterized in that ineach case one fragment of the elongated oligonucleotides is detected inthe mass spectrometer.
 17. The method as recited in claim 16,characterized in that the fragment of the elongated oligonucleotide isproduced by digestion with one or several exo- or endonucleases.
 18. Themethod as recited in claim 17, characterized in that the producedfragments have a single positive or negative net charge for betterdetectability in the mass spectrometer.
 19. The method as recited inclaim 1, characterized in that the detection of the elongatedoligonucleotides is carried out and visualized by means of matrixassisted laser desorption/ionization mass spectrometry (MALDI) or usingelectron spray mass spectrometry (ESI).
 20. The method as recited inclaim 1, wherein the polymerases are heat-resistant DNA-polymerases. 21.The method as recited in claim 1, wherein SNPs are also detected andvisualized in addition to the DNA methylation.
 22. The method as recitedin claim 1, wherein the used nucleotides are terminating (type C 2)and/or chain-elongating nucleotides (type C 1).
 23. The method asrecited in claim 1, wherein the nucleotides (type C 1 and C 2) areselected from a group comprising either the nucleobases A, T and C orelse the bases G and A and T.
 24. The method as recited in claim 1,characterized in that the amplification of several DNA segments iscarried out in one reaction vessel.
 25. The method as recited in claim1, step a, wherein the genomic DNA is obtained from a DNA sample,sources of DNA comprising, e.g., cell lines, blood, sputum, stool,urine, cerebral-spinal fluid, tissue embedded in paraffin, histologicobject slides, and all possible combinations thereof.
 26. The method asrecited in claim 1, characterized in that the methylation analyses ofthe upper and lower DNA strands are carried out in one experiment.